In-vivo reactive species imaging

ABSTRACT

The disclosure features methods that include: administering to a subject a composition that includes particles, where each one of the particles features at least one targeting group that binds to a structural entity in the subject and at least one reacting group that reacts chemically with a reactive oxygen species in the subject, and where the particle emits luminescence when the reaction occurs; detecting the luminescence emission from the particles; and displaying an image of the subject showing locations of at least some reactive oxygen species in the subject based on the detected luminescence.

TECHNICAL FIELD

This disclosure relates to imaging of reactive oxygen species, includingimaging in living subjects.

BACKGROUND

Reactive oxygen species (ROS) are implicated in a variety of biologicalfunctions. For example, intracellular changes in ROS can influence cellsignaling by thiol oxidation within target proteins, resulting inchanges that affect the structure-function properties of the proteins.Oxidation by ROS has also been attributed to activating kinases andinhibiting phosphatases, leading to enhancement of phosphorylationcascades. Conformational changes due to oxidation by ROS can also resultin changes in protein stability, protein-protein and protein-DNAinteractions, and subcellular localization. Redox regulation, in whichROS play a role, has been attributed to various signaling pathways andmay affect cellular responses such as transcription regulation,proliferation, migration, metabolism, survival and inflammatoryresponse.

SUMMARY

Redox signaling in cells depends on the balance between in-vivoproduction of ROS, and scavenging of excess ROS by intracellularantioxidant species ROS sources include NADPH oxidases and mitochondria(e.g., the mitochondrial electron transport chain). NADPH oxidasesproduce superoxide (O₂), which is rapidly converted to hydrogen peroxide(H₂O₂) by superoxide dismutases. When a subject is affected by certainpathophysiological conditions such as cancer and inflammatory disease,aberrant redox signaling often accompanies other symptoms of thecondition. Aberrant redox signaling, in turn, may be the result ofdisruption of the balance between ROS production and ROS scavenging byantioxidant species. In particular, enhanced production of ROS indiseased tissue and/or reduced production of antioxidant species such assuperoxide dismutases, catalase, glutathione peroxidase, thioredoxin,peroxiredoxin, and/or the glutathione family, can lead to suchimbalances. As a result, localization and quantification of ROS in-vivocan provide important diagnostic information for identifying variouscancers and inflammatory conditions.

In general, in a first aspect, the disclosure features methods thatinclude: administering to a subject a composition that includesparticles, where each one of the particles features at least onetargeting group that binds to a structural entity in the subject and atleast one reacting group that reacts chemically with a reactive oxygenspecies in the subject, and where the particle emits luminescence whenthe reaction occurs; detecting the luminescence emission from theparticles;

and displaying an image of the subject showing locations of at leastsome reactive oxygen species in the subject based on the detectedluminescence.

Embodiments of the methods can include any one or more of the followingfeatures.

Administering the composition can include injecting the particles in abody of the subject. The subject can be a living human. The subject canbe a living mammal (e.g., a mouse, a rat). The subject can be a livingbird, a living amphibian, or a living fish.

The methods can include detecting luminescence emission at a wavelengthof 500 nm or more (e.g., 600 nm or more, 700 nm or more). The methodscan include displaying the image of the subject based on unfilteredemitted radiation from the subject, where the unfiltered emittedradiation includes the luminescence emission.

The at least one targeting group can include at least one antibody. Thereactive oxygen species can include singlet oxygen and/or hydroxideradical and/or hypochlorous acid and/or superoxide radical and/or nitricoxide and/or hydrogen peroxide.

Each one of a first subset of the particles can include a firsttargeting group and a first reacting group, and each one of a secondsubset of the particles can include a second targeting group and asecond reacting group, where the first and second targeting groups aredifferent. The first and second targeting groups can bind to differentstructural entities in the subject. The first and second targetinggroups can include different antibodies.

Each one of the first subset of the particles can emit luminescence at afirst central wavelength, and each one of the second subset of theparticles can emit luminescence at a second central wavelength differentfrom the first central wavelength. Each of the first and second centralwavelengths can be greater than 500 nm.

The first and second reacting groups can be the same.

Embodiments of the methods can also include any of the other features orsteps disclosed herein, in any combination, as appropriate.

In another aspect, the disclosure features compositions that include asuspension medium and a plurality of particles, where each one of theplurality of particles features: a core that includes at least onereacting group that reacts chemically with a reactive oxygen species inthe subject, and at least one luminescent agent that emits luminescencein response to the reaction of the at least one reacting group; a firstcoating material encapsulating the core; and a second coating materialencapsulating the first coating material and including at least onetargeting group that binds to a structural entity in the subject.

Embodiments of the compositions can include any one or more of thefollowing features.

The core can include at least one of latex and polystyrene. The firstcoating material can include aminodextran and the second coatingmaterial can include dextran aldehyde.

The at least one targeting group can include at least one antibody. Theat least one luminescent agent can include at least one lanthanideelement. The at least one lanthanide element can include at least one ofeuropium and terbium. The at least one reacting group can includethioxene or a thioxene derivative.

The plurality of particles can include: a first subset of particlesfeaturing a first targeting group, a first luminescent agent, and afirst reacting group; and a second subset of particles featuring asecond targeting group, a second luminescent agent, and a secondreacting group, where the first and second targeting groups aredifferent. The first and second targeting groups can include differentantibodies.

The first and second luminescent agents can be different. The first andsecond luminescent agents can include different lanthanide elements.

Any one or more of the compositions disclosed herein can be included ina kit for imaging reactive oxygen species in a living subject. Thereactive oxygen species can include at least one member selected fromthe group consisting of singlet oxygen, hydroxide radical, hypochlorousacid, superoxide radical, nitric oxide, and hydrogen peroxide.

Embodiments of the compositions can also include any of the otherfeatures disclosed herein, in any combination, as appropriate.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the subject matter herein, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features and advantages willbe apparent from the description, drawings, and claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a particle for in-vivo detection ofreactive species.

FIG. 2 is a schematic diagram of a kit that includes particles forin-vivo detection of reactive species.

FIG. 3 is a graph showing luminescence signals for reactive oxygenspecies detected in vitro by a plurality of particles.

FIG. 4 is a series of images showing measured luminescence signalscorresponding to reactive oxygen species in two groups of murinesubjects.

FIG. 5A is a series of images showing measured luminescence signals fromthe same murine subjects as in FIG. 4, obtained over a period of 1 hour.

FIG. 5B is a graph showing luminescence signals measured from lungtissue for the same murine subjects as in FIG. 4.

FIG. 5C is a graph showing luminescence signals measured from livertissue for the same murine subjects as in FIG. 4.

FIG. 6A is a series of images showing measured luminescence signals fromantibody-conjugated particles in two groups of murine subjects.

FIG. 6B is a graph showing luminescence signals measured for the samemurine subjects as in FIG. 6A at two different times.

FIG. 7A is a series of images showing measured luminescence signals fromantibody-conjugated particles in two groups of murine subjects.

FIG. 7B is a graph showing luminescence signals measured from lung andliver tissue for the same murine subjects as in FIG. 7A.

FIG. 8A is a series of images showing measured luminescence signals fromantibody-conjugated particles in fasting and non-fasting murinesubjects.

FIG. 8B is a graph showing luminescence signals measured from lung andliver tissue for the same murine subjects as in FIG. 8A.

FIG. 9A is at set of images showing luminescence signals from particleswith terbium or europium luminescent agents in a group of murinesubjects.

FIGS. 9B and 9C are graphs showing luminescence signals measured fromthe left and right thighs, respectively, of the same murine subjects asin FIG. 9A.

FIG. 10 is a schematic diagram of the chemical structure of C-28thioxene dye.

FIG. 11 is a schematic diagram of the chemical structure of a chelatedlanthanide element such as europium or terbium.

FIG. 12 is a schematic diagram of the chemical structures of examples ofdi-ketonate ligands that can be used to chelate lanthanide elements.

FIG. 13 is a schematic diagram of the chemical structures of examples ofphenanthroline ligands that can be used to chelate lanthanide elements.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Reactive oxygen species (ROS) have been detected using combinations ofdifferent types of particles. In particular, combinations of donor andacceptor beads have been used as in-vitro ROS sensors. Typically, thedonor and acceptor beads are brought into proximity by an analyte in asandwich where illuminating the donor particles at the wavelength of thesensitizer generates singlet oxygen or other ROS. ROS thus generatedchemically react with an electron rich olefin or reactive oxygen sensorin the acceptor beads, which emit light following the reaction. Theemitted light from the acceptor beads can be transferred to anappropriate acceptor in a FRET manner which can be imaged or read in theplate reader.

In general, the acceptor beads are able to sense ROS only within afinite distance as ROS decay rapidly within their lifetime ofapproximately 2 microseconds. For example, singlet oxygen typicallytraverses distance of only about 200 nm before it decays. As a result,efficient excitation of an acceptor bead by a donor bead typicallyoccurs when the acceptor and donor beads approach relatively closely(e.g., within a few hundred nanometers). Moreover, the use of donor andacceptor beads can generate autofluorescence in tissue. Autofluorescenceis typically observed as emission of light across a relatively wide bandof visible wavelengths. Autofluorescence functions as a backgroundsignal against which fluorescence emission signals from acceptor beadsthat indicate the presence of ROS are distinguished.

The present disclosure features compositions of particles for detectingand imaging ROS in-vivo that do not include both donor and acceptorbeads. Instead, particles in the compositions disclosed herein featuretargeting groups that bind to structural entities in the subject's body,reacting groups that react with ROS to generate radiation, andluminescent agents that absorb the generated radiation and emitluminescence. The luminescence can be detected (e.g., imaged) to showspatial location and quantification of the ROS in the subject's body.

For purposes of this disclosure, reactive oxygen species (ROS) includesingle oxygen, hydroxide radical, hypochlorous acid, superoxide radical,nitric oxide, hydrogen peroxide, and other products of mediation byoxidases such as myeloperoxidase and horseradish peroxidase.

FIG. 1 is a schematic diagram of a particle 100 that includes a core102, a first coating material 104, and a second coating material 106.Particle 100 reacts with ROS in a subject's body and emits luminescence.Core 102 typically corresponds to a bead formed of one or morebiologically compatible materials. In some embodiments, for example,core 102 can be formed of latex, polystyrene, acrylic acid, amides, andother materials bearing unsaturated groups such as olefinic and/oracetylenic groups that can be polymerized, e.g., under emulsiveconditions.

First coating material 104 and second coating material 106 typicallyeach include one or more polysaccharides. In general, a wide variety ofdifferent polysaccharides can be used alone or in combination to formcoating materials 104 and 106. For purposes of this disclosure, suitablepolysaccharides that can be used to form coating materials 104 and 106include, carbohydrates that include three or more non-modified ormodified monosaccharide units, such as, e.g., dextran, starch, glycogen,inulin, levan, mannan, agarose, galactan, carboxydextran and/oraminodextran. Examples of polysaccharides include, but are not limitedto, dextran, starch, glycogen, polyribose, and functionalizedderivatives thereof.

Core 102 also includes at least one reacting group that reacts with oneor more ROS to generate radiation. In general, a variety of differentmaterials can be used as reacting groups in core 102. Examples ofsuitable reacting groups include thioxene derivatives, electron richolefins in dioxetanes, dienes, and aromatics, either as isolated speciesor conjugated with an acceptor dye moiety. Reacting groups are furtherdisclosed in the following U.S. Patents, the entire contents of each ofwhich is incorporated herein by reference: U.S. Pat. Nos. 6,406,913;6,692,975; 6,916,667; and 6,406,667.

Core 102 also includes at least one luminescent agent. The at least oneluminescent agent absorbs radiation emitted by the reacting groups, andemits luminescence. Typically, the central wavelength of the emittedluminescence is larger than the central wavelength of the absorbedradiation, where the central wavelength refers to the center of thefull-width at half-maximum of the distribution of the absorbed oremitted radiation. For example, in some embodiments, the centralwavelength of the emitted luminescence is 500 nm or more (e.g., 550 nmor more, 600 nm or more, 650 nm or more, 700 nm or more, 750 nm or more,800 nm or more, 850 nm or more).

A variety of different luminescent agents can be used in core 102. Insome embodiments, for example, the luminescent agents can include one ormore lanthanide elements (e.g., one or more of lanthanum, cerium,praseodymium, neodymium, promethium, samarium, europium, gadolinium,terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium).Among these elements, europium and terbium are particularly advantageousfor use as luminescent agents.

Second coating material 106 includes at least one targeting group thatbinds to a structural entity in the subject. Structural entities in thesubject typically include proteins, antibodies, nucleic acids, aptamers,truncated forms of antibodies such as Fabs, cysbodies, and diabodies,and other chemical structural moieties that are of interest. Structuralentities that are targeted by the at least one targeting group include,but are not limited to, poly(amino acids), i.e., polypeptides andproteins, polysaccharides, nucleic acids, and combinations thereof. Suchcombinations include components cells, such as chromosomes, genes,mitochondria, nuclei, and cell membranes. In general, the poly(aminoacids) have a molecular weight of between 5,000 and 5,000,000 Daltons.Structural entities can also include oligonucleotides andpolynucleotides such as m-RNA, r-RNA, t-RNA, DNA (double stranded (“ds”)or single stranded (“ss”)), RNA (ds or ss), DNA-RNA duplexes, etc. An“oligonucleotide” is a single stranded polynucleotide including asynthetic polynucleotide. Oligonucleotides typically include a sequenceof 10 to 100 nucleotides. A “polynucleotide” is a compound orcomposition which is a polymeric nucleotide having in the natural stateabout 50 to 500,000 or more nucleotides. Polynucleotides include nucleicacids from any source, such as DNA (dsDNA and ssDNA) and RNA (dsRNA andssRNA), t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNAand RNA, DNA-RNA hybrids and/or mixtures thereof, genes, chromosomes,plasmids, and genomes of biological material.

Targeting groups that bind to the structural entities include groupsthat include a structural feature or region that specifically binds to,and is therefore complementary with, one or more of the structuralentities in the subject. A variety of different targeting groups can bepresent in second coating material 106, including one or more naturallyoccurring and/or synthetic molecules, e.g., thyroxine binding globulin,steroid-binding proteins, antibodies, Fab fragments or otherantigen-binding fragments of antibodies, enzymes, lectins, nucleicacids, repressors, oligonucleotides, protein A, protein G, avidin,streptavidin, biotin, complement component C1q, DNA binding proteins,and RNA binding proteins. The targeting groups can be members (togetherwith the structural entities in the subject) of an immunological pairsuch as antigen-antibody or haptenantibody, operator-repressor,nuclease-nucleotide, biotin-avidin, lectin-polysaccharide,steroid-steroid binding protein, drug-drug-receptor, hormone-hormonereceptor, enzyme-substrate, IgG-protein A, and/or oligo- orpolynucleotide-complementary oligo- or polynucleotide.

In general, one of first coating material 104 and second coatingmaterial 106 includes amine functional groups, and the other coatingmaterial includes amine-reactive functional groups. For example, thepolysaccharide(s) of first coating material 104 can be covalentlycoupled to core 102 by reaction between amine-reactive functional groupsof the core (i.e. carboxyl groups) and amine groups of thepolysaccharide(s). The polysaccharide(s) of second coating material 106can be covalently coupled to the polysaccharides of first coatingmaterial 104 by reaction between the amine functional groups of thepolysaccharide(s) of the first coating material 104 and amine-reactivefunctional groups of the polysaccharide(s) of second coating material106. Conversely, in some embodiments, core 102 has amine functionalgroups that react with amine-reactive functional groups of thepolysaccharide(s) of first coating material 104. The polysaccharide(s)of second coating material 106 also include amine-reactive functionalgroups.

Particle 100 in FIG. 1 includes two coating materials 104 and 106. Moregenerally, however, more than two coating materials can be used. In someembodiments, for example, one or more additional coating materials canbe applied to particle 100. The additional coating materials cangenerally include any of the materials disclosed herein in connectionwith coating materials 104 and 106. For example, the additional coatingmaterials can each include one or more polysaccharides.

Additional coating materials can also include any one or more of theadditional components disclosed herein. For example, in someembodiments, additional coating materials can include one or moreadditional targeting groups that bind to specific structural entitieswithin the subject. The targeting groups can include any of thetargeting groups disclosed above. Providing additional targeting groups,either in second coating material 106 or in additional coatingmaterials, can allow particle 100 to bind to a wide variety ofstructural entities.

As an example, the following describes a preparative method for aplurality of particles 100 that include a first coating material 104featuring aminodextran (AmDex) and a second coating material featuringdextran aldehyde (DexAl), with specific reactive groups and luminescentagents. However, it should be understood that this method is merelyillustrative of only certain embodiments, and can readily be adapted toinclude any of the first and second coating materials, reactive groups,luminescent agents, and targeting groups disclosed herein.

Carboxylated latex or polystyrene particles were obtained in theirnative, uncoated state. To introduce the reacting group(s) andluminescent agent(s), the particles were heated to slightly below theirglass transition temperature, rendering them porous. A quantity ofthioxene dye (C-28 thioxene, shown in FIG. 10), which functions as thereacting groups, was added to the beads. Further, chelated europiumand/or terbium was added to the beads (structure shown in FIG. 11, withLa=Eu, Tb). The particles had a diameter of between 190 nm and 210 nm,and a surface carboxy-group concentration of about 60,000 per particle.

To introduce C-28 thioxene and chelated Eu or Tb, 5 mL Seradyncarboxylated polystyrene particles (diameter ˜200 nm) at a concentrationof 100 mg/mL were added to a 50 mL flask, along with 75 μL NaOH at 1 M,7.0 mL 2-ethoxylethanol, and 20.5 mL water. The particle suspension washeated to 80° C. using an oil bath with constant stirring. To a glassvial were added 100 mg C-28 thioxene, 135 mg chelated Eu or Tb (e.g.,Tb(XTA)₃.Phen), and 11 mL 2-ethoxyethanol. This solution was added tothe stirred particle suspension at 80° C. Heating and stirring continuedfor another 25 minutes. The mixture was then cooled to room temperaturewith continued stirring.

The suspension was filtered, and 200 mL EtOH 10% at pH >10 was added tothe filtrate and mixed. The combined suspension was centrifuged for 20minutes at 15,000 rcf using an Eppendorf 5417C centrifuge. Aftercentrifugation, the liquid was decanted and the particles re-suspendedin 10% EtOH at pH >10, and briefly sonicated.

Chelated Eu and/or Tb was prepared in a one-step synthesis. Theprocedure below discloses the preparation of chelated Tb, but can alsobe used for chelated Eu with minor modifications. 3.72 grams (10 mmol)of TbCl₃.6H₂O in 30 mL water were added to a 250 mL Erlenmeyer flask. 90mL of absolute EtOH was added to the solution and mixed to achieve ahomogeneous distribution. Next, 6.48 g (30 mmol) of1-benzoyl-3,3,3-trifluoroacetonate (BTA) was added to the solution withstirring for 1 hour to complete dissolution. Then, 6.6 mL of 5 M NaOH(33 mmol) was added slowly, maintaining the pH between 6 and 7. Thesolution was heated to between 50° and 60° C. for 1 hour with stirring.

Meanwhile, 1.80 g (10 mmol) of 1,10-phenanthroline was dissolved in 30mL absolute EtOH, and was added to the still-warm solution followingheating. The combined solution was stirred for 1 hour without furtherheating, forming a precipitate. The mixture was allowed to cool duringstirring. Stirring continued at high speed to prevent clumping of theproduct. 150 mL of water was added to the mixture, which was then mixedthoroughly for another hour. The slurry was then cooled for 1 hour in arefrigerator at about 4° C. Solids were filtered from the mixture usinga Buchner funnel, and were washed 3 times with 50 mL water. The solidchelated Tb product was transferred to a Petri dish and allowed to airdry for several days at room temperature, or dried in a vacuumdessicator overnight.

In general, a variety of different diketonates can be used in theprocedure described above (e.g., with different substituentscorresponding to aromatic ring R in FIG. 11). Examples of suitablediketonates are shown in FIG. 12. Further, a variety of differentphenanthrolines can be used in the procedure described above (e.g., withdifferent substituents corresponding to group R′). Examples of suitablephenanthrolines are shown in FIG. 13.

The preparation of suitable thioxene dyes and chelated lanthanideelements, including chelated europium and terbium, is also described forexample in the following patents, the contents of each of which isincorporated by reference herein: U.S. Pat. Nos. 5,340,716; 6,251,581;6,406,913; 6,692,975; 6,916,667; 7,179,660.

Next, the particles (i.e., cores 102 with reacting groups andluminescent agents added) were coated with AmDex molecules (havingmultiple amino groups per AmDex molecule) using carbodiimide (EDAC)conjugation chemistry. The presence of opposite charges on the particlesurface (—COO⁻) and on the AmDex (—N⁺H₃) attract each other and in thepresence of a relatively large excess of AmDex, the particle surface israpidly and effectively covered by AmDex molecules, even in the absenceof EDAC. In this fashion, the polysaccharide spontaneously associateswith the particles, leading to an increased concentration of aminogroups near to the surface of the particles which in turn improves theefficiency of the EDAC conjugation method. Adding EDAC at this pointactivates the —COOH groups on the particle, which subsequently reactwith —NH₂ groups on AmDex leading to the formation of a chemicallystable amide bond. Only a fraction of the amino groups from the AmDexmolecule are involved in forming the covalent amide bond and theremaining amino groups are available for reactions with various othergroups (e.g., aldehyde groups or carboxyl groups). The coated particleshad a diameter of between 240 nm and 270 nm, with 1 amino group forevery 12 saccharides.

To coat the particles, 300 mg of particles in suspension were spun downand the liquid removed. The particles were re-suspended in 100 mM2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.0 to a concentrationof 20 mg/L, for a total volume of 15 mL. To this suspension was added 15mL of amino-dextran at a concentration of 8 mg/mL in water, 5 mL EDAC ata concentration of 80 mg/mL in water. After mixing, the mixture wasincubated for 2 hours at room temperature with agitation. The particleswere then washed twice with 100 mM MES at pH 6.0 by centrifugation at15,000 rcf for 20 minutes. The particles were washed once with 0.1 N HCland finally collected in 12.5 mL of 100 mM MES at pH 6.0 and sonicated.

The particles were subsequently coated with a second polysaccharidelayer by reacting the particles with a relatively large excess ofdextran aldehyde (DexAl) having multiple aldehyde groups per DexAlmolecule. This spontaneous reaction is a form of spontaneous associationof the polysaccharide layers. A fraction of the aldehyde groups on aDexAl molecule react with amino groups on the AmDex layer formingshiff-base, which then can be reduced with a mild reducing agent such ascyanoborohydride (NaBH₃CN) to form a chemically stable carbon-nitrogenbond. Since the reduction of free aldehyde groups by NaBH₃CN isnegligible, the resulting coated particles have reactive aldehyde groupson the surface and can react with amino group containing molecules (suchas antibodies or other proteins or further AmDex molecules). The coatedparticles had a diameter of 260 nm to 310 nm, with 1 aldehyde group forevery 16 saccharides (approximately 30,000 to 50,000 per particle).

To apply the second polysaccharide layer, 5 mL of the particlesuspension (concentration 20 mg/mL) was added to a glass vial, alongwith 0.8 mL dextran aldehyde solution (concentration 50 mg/mL), 4.2 mLof 0.1 M MES at pH 6.0, and 0.5 mL NaBH₃CN at a concentration of 80mg/mL in water (freshly prepared). After mixing, the vial was closedwith a screw cap and incubated at 37° C. overnight. After equilibratingto room temperature, the particles were washed twice with 0.1 M MES atpH 6.0 by centrifugation at 15,000 rcf for 20 minutes. The particleswere then sonicated and collected in approximately 4 mL of 0.1 M MES atpH 6.0.

One or more targeting groups were then conjugated to the secondpolysaccharide layer. The conjugation was performed by a reductiveamination method, in which purified antibody in native form (notmodified) was incubated with the particles in presence of NaBH₃CN for acertain period of time, preferably at room temperature or at 37° C. Theremaining free aldehyde groups were capped (various molecules can beused for this purpose, for example carboxymethyl oxime orcarboxymethoxylamine, etc.)

Conjugation typically occurs best when the antibody concentration is atleast 1 mg/mL (for conjugation of 1-2 mg of particles) or 0.53 mg/mL(for conjugation of 2.5 mg or more of particles). Antibody solutions canbe concentrated, for example, using an iCON Concentrator (available fromThermoFisher Scientific, Waltham, Mass.). Typically, the antibodies arenot in an amine-based buffer (e.g., Tris, glycine, bicine, tricine). Ifbuffer exchange is undertaken, the buffer solution is generally replacedby a neutral or slightly alkaline buffer such as PBS or carbonate bufferat a pH of about 8.0. In addition, antibody solutions used forconjugation typically do not include protein- or peptide-basedstabilizers (e.g., BSA, gelatin) or glycerol. Protein stabilizers can beremoved using PhyTip affinity columns (available from PhyNexus, SanJose, Calif.) or a liquid handling system such as the JANUS automatedworkstation (available from PerkinElmer, Waltham, Mass.). Dialysis canbe used to remove glycerol.

When conjugating particles, the ratio of antibody to mass of particlesis an important parameter. Typical coupling ratios (e.g., mass ofparticles to mass of antibody) are either 10:1 (for 1-2 mg of particles)or 50:1 (for 2.5 mg of particles or more). For example, for 5 mg ofparticles, 0.1 mg of antibody is typically used.

The following procedure was used to conjugate 5 mg of particles, using a50:1 coupling ratio with an antibody. The antibody solution usedtypically has a concentration of 0.53 mg/mL or more. The particles werefirst washed in a 1.5 mL Eppendorf tube in 250 μL of water, and then 250μL of PBS was added. The suspension was centrifuged at 16,000 g (ormaximum speed) for 15 minutes and the supernatant liquid discarded usinga pipet tip.

A fresh working solution of NaBH₃CN (obtained in powder form fromSigma-Aldrich, St. Louis, Mo.) at a concentration of 400 mM in water wasprepared by adding 25 mg of NaBH₃CN powder to 1 mL of water. To theEppendorf tube containing the washed particles were added 0.1 mg ofantibody, a volume of 100 mM Hepes pH 7.4, to achieve a final reactionvolume of 200 μL, 1.25 μL of 10% Tween-20 (obtained from Thermo-FisherScientific), and 10 μL of the aqueous NaBH₃CN solution. The suspensionwas incubated for 18-24 hours at 37° C. using a rotary shaker at 6-10RPM.

Next, a fresh 65 mg/mL solution of carboxymethoxylamine (CMO) (obtainedfrom Sigma-Aldrich) in 800 mM NaOH was prepared, and 10 μL of the CMOsolution was added to the suspension, which was incubated for anadditional 1 hour at 37° C. using a rotary shaker at 6-10 RPM. The CMOsolution blocks unreacted sites on the particles.

The conjugated particles were then washed. First, the particles werecentrifuged for 15 minutes at 16,000 g (or maximum speed) at 4° C. Thesupernatant was removed using a micropipette and the dehydrated particlepellet was resuspended in 1 mL of 100 mM Tris-HCl pH 8.0 (using about200 μL per mg of particles). The particle suspension was brieflysonicated (10 short pulses of 1 second using a probe sonicator) toensure the particles were not aggregated at a sonicator power ofapproximately 20% or maximum power. The suspension was then centrifugedfor 15 minutes at 16,000 g or maximum speed at 4° C., and thesupernatant removed.

The foregoing washing steps were then repeated. Following the lastcentrifugation, the particles were re-suspended at a concentration of 5mg/mL in a storage buffer (1 mL of PBS and 0.05% Proclin-300 as apreservative). The suspension was vortexed, briefly spun down, andsonicated using 10 short pulses of 1 second using about 20% of maximumsonication power.

Conjugated particles were stored in opaque vials at a temperature of 4°C. Prior to use, the suspensions were vortexed again to counteractsettling during storage.

To conjugate quantities of particles larger than 5 mg, the foregoingprocedure was adapted to use a 50 mL 3118 Oak Ridge centrifuge(available from Thermo Fisher Scientific) with a maximum volume in eachtube of less than about 30 mL to allow proper centrifugation.Centrifugation steps were performed in a Sorvall RC-5B centrifuge(Thermo Fisher Scientific) at 16,000 g for 40 minutes at 4° C.

In general, the particle coupling procedure disclosed herein can beoptimized (e.g., to increase the number of antibodies bound perparticle) by reducing the reaction volume, as coupling efficiencytypically increases with particle concentration. Increased particleconcentrations, up to 100 mg/mL, can be prepared by adding a smallervolume of a more concentrated buffer solution to the particles.

The procedure can also be optimized by increasing the ratio of antibodyto particles, as coupling efficiency typically increases with antibodyconcentration. In general, the antibody stock solution should besufficiently concentrated so that only a small additional volume of theantibody solution is added to increase the antibody concentrationwithout significantly diluting the particle concentration. For example,a 10:1 ratio of particles:antibody, when implemented at a particleconcentration of 25 mg/mL, can improve conjugation efficiency.Increasing the particle concentration to 75 mg/mL while maintaining aratio of 50:1 will typically involve an antibody concentration of 1.7mg/mL. Increasing the relative proportion of antibody to a ratio of 25:1will involve an antibody concentration of 3.4 mg/mL, which is close tothe practical upper limit. Other buffers can also be used to optimizethe foregoing procedure (e.g., 100 mM sodium phosphate at pH 8.0).

Additional preparative methods and materials are disclosed, for example,in the following patents and patent publications, the entire contents ofeach of which is incorporated herein by reference: U.S. Pat. Nos.5,340,716; 6,251,581; 6,406,913; 6,692,975; 6,916,667; 7,179,660; andPCT Patent Publication No. WO 2001/067105.

The particles disclosed herein can be introduced into a living subjectusing a variety of methods, including injection, e.g., subcutaneousinjection, intravenously, intratracheally, intranasally). Typically, theparticles are introduced in proximity to a region of interest in thesubject, e.g., near the site of a suspected tumor or tissueinflammation. The targeting groups are selected so that the particlesbind selectively to certain structural entities, e.g., entities that arepresent in the region of interest. The targeting of specific structuralentities within the subject allows the particles to function assensitive diagnostic reporters for reactive species, e.g., reactiveoxygen species in the subject's body.

The particles can be used detect and image reactive species such as ROSin a variety of different subjects. In general, the particles can beused for in-vivo detection and imaging in humans, in mammals (including,but not limited to, mice, rats, dogs, and cats, and more generally, anylaboratory mammalian subject), in birds, reptiles, amphibians, and fish.The particles can also be used for in-vitro detection and imaging oftissue samples and/or biological fluids extracted from any of theforegoing subjects.

After introducing the particles into the subject's body, luminescenceemitted by the particles is imaged using a detector (e.g., a CCD-baseddetector or a CMOS-based detector). In combination with light reflectedfrom the surface of the subject, the emitted luminescence shows wherereactive species, such as reactive oxygen species, are located withinthe subject's body. In some embodiments, the detected luminescence canbe displayed directly to a viewer without filtering the luminescence. Incertain embodiments, the detected luminescence can be overlaid on animage of the subject's body. Both of these display modalities can yieldimages that localize the ROS relative to other features of the subject'sbody.

The intensity and distribution of luminescence detected can also providequantitative information about ROS in the subject's body. For example,by spatially integrating portions of the luminescence image of thesubject and correlating the integrated intensity with calibration datathat relates light intensity to ROS concentration, the concentration ofROS in the integrated region of the subject's body can be estimated.Quantitative estimates of ROS concentrations can provide additionalinformation for purposes of diagnosing diseases in the subject.

In some embodiments, more than one type of particle can be administeredto the subject. For example, the plurality of particles that isadministered can include a first subset of particles with one type oftargeting group and a second subset of particles with a second type oftargeting group. By providing particles with different targeting groups,the particles can bind to different structural entities within thesubject, thereby providing diagnostic ROS information about multipleregions of the subject's body.

Additionally, or alternatively, the first subset of particles caninclude a first luminescent agent and the second subject of particlescan include a second luminescent agent. Different luminescent agentstypically emit luminescence at different central wavelengths. Thus,subsets of particles with different luminescent agents can be used toprovide information about different regions of a subject's body.

For example, in some embodiments, the first subset of particles caninclude both targeting groups and luminescent agents that are differentfrom the targeting groups and luminescent agents of the second subset ofparticles. Providing subsets of particles with these features yieldstwo-fold selectivity in targeting particular structures or regionswithin a subject's body. First, the two subsets of particles bind todifferent structural entities in the subject on account of theirdifferent targeting groups, thereby providing a first mechanism fordistinguishing the different entities. Second, luminescence from thedifferent subsets of particles occurs at different central wavelengths.Accordingly, by filtering and/or selectively detecting luminescence atcertain wavelengths, ROS in the different structural entities can beseparately identified and quantified.

The particles disclosed herein can be provided in a diagnostic kit forclinical use. FIG. 2 is a schematic diagram of a kit 200 that includes ahousing (e.g., a wrapper) 202. Enclosed within the housing is acontainer 204 that includes a plurality of particles 100. Optionally,kit 200 can include a second container 206 that includes, for example,one or more buffer solutions for introducing the particles into asubject's body (e.g., via injection). In some embodiments, particles 100are provided in container 204 in an unsuspended (e.g., dry) form. Priorto administering the particles, the particles are suspended in asolution provided in second container 206. In certain embodiments,particles 100 are provided in container 204 already suspended in aphysiologically-compatible solution. The particles can either beadministered directly to the subject, or diluted to a desiredconcentration (e.g., in a buffer or other solution) before they areadministered to the subject.

The particles disclosed herein provide a number of important advantages.For example, the particles emit luminescence, which is detected and usedto localize and quantify ROS in the subject. In contrast tofluorescence, luminescence typically occurs toward the red edge of thevisible region of the spectrum, and in the near-infrared region. As aresult, particles which emit luminescence are better suited to act asreporters for ROS in deep tissue applications (e.g., where ROS arelocated in tissues more than about 2 mm below the skin surface) becausetissue-dependent scattering of radiation is not as strong at longerwavelengths. Thus, the particles disclosed herein provided significantlyimproved diagnostic sensitivity for ROS located in deep tissues relativeto fluorescence-based particles and reporters.

In addition, fluorescence-based reporters are typically accompanied byautofluorescence in the subject's tissue, which functions as abackground signal against which fluorescence emission due to detectionof ROS is distinguished. In contrast, the luminescence-based particlesdisclosed herein do not typically excite, and are not typicallyaccompanied by, significant autofluorescence in the subject's tissue. Asa result, the luminescence emission typically occurs against anessentially “dark” background; removal of autofluorescence contributions(e.g., by sophisticated analysis algorithms) is generally not required.In some embodiments, for example, the detected luminescence can be usedto visualize and quantify ROS in the subject's body without anyfiltering of the signal measured by the detector.

Further, the particles disclosed combine selective binding, reactionwith ROS, and luminescent reporting of ROS in a single particle.Conventional two-particle methods for detecting ROS, which involve bothdonor and acceptor particles, provide acceptable signals only when donorand acceptor particles approach one another closely enough. In contrast,the particles disclosed herein do not rely on the distance betweenparticles to detect ROS. Instead, each particle includes a reactinggroup that reacts with ROS, and a luminescent agent that emitsluminescence in response to energy transfer from the reacting group.Because the luminescent agent and reacting group are located on the sameparticle, the energy transfer is efficient.

Although the methods and compositions disclosed above are directed toparticles for detection of reactive oxygen species in vivo, moregenerally the methods and compositions can also be applied to thedetection of other reactive species in vivo. For example, the methodsand compositions can be applied to in vivo detection of reactivenitrogen species (e.g., NO, NO radical, and peroxynitrite anion ONOO⁻).To detect reactive nitrogen species, particles 100 can include one ormore reacting groups that react with reactive nitrogen species, emittingradiation that is absorbed by the luminescent agent(s) to causeluminescence emission. Suitable reacting groups include, for exampleDAF-FM (available from Thermo Fisher Scientific), which is a reagentthat is used to detect and quantify low concentrations of nitric oxide(NO). DAF-FM is essentially non-fluorescent until it reacts with NO toform a fluorescent benzotriazole. DAF-FM fluorescence can be detectedusing a variety of different techniques and devices, including flowcytometers, microscopes, fluorescent microplate readers andfluorometers, and imaging systems such as the IVIS and FMT systems,available from from Perkin Elmer.

Additional reacting groups and substrates therefor are disclosed, forexample, in Xiaoqiang Chen et al., “Fluorescent and luminescent probesfor detection of reactive oxygen and nitrogen species,” Chem. Soc. Rev.40: 4783-4804 (2011), the entire contents of which are incorporatedherein by reference.

Examples

The subject matter disclosed herein is further described in thefollowing examples, which are not intended to limit the scope of theclaims.

To evaluate the efficiency with which various ROS are detected using theparticles disclosed herein, quantities of HOCl, hydroxide radical,superoxide, nitric oxide, and hydrogen peroxide were prepared in vitro.Each of these in vitro species was treated with particles 100, whichwere prepared as described above and included latex cores, a firstcoating of aminodextran, and a second coating of dextran aldehyde. Athioxene dye and chelated europium were introduced into the latex coresbefore coating.

FIG. 3 is a graph showing a comparison of luminescence signal/backgrounddetected for each of the different ROS. Each of the species was reliablydetected, with hydroxyl radical and superoxide having the highest signalabove background.

To evaluate the efficiency of in vivo murine detection of ROS, a sampleof BALB/cJ mice subjects were divided into two groups. The first groupwas challenged by intracheal delivery of 50 μL of 1 mg/kglipopolysaccharide (LPS) at a concentration of 1 mg/kg i.n. to induce aninflammatory response. The second group, functioning as the control, waschallenged with phosphate buffered saline (PBS). A 10 μg quantity ofparticles (prepared as described above, with aminodextran and dextraaldehyde coatings, thioxene dye and chelated europium introduced intothe latex core) were introduced into both lung and liver sites viasubcutaneous injection 3 hours after the LPS challenge. Luminescenceemission from the injected particles was imaged as a function of timefollowing the injection of the particles. FIG. 4 shows images of control(left side) and LPS-challenged (right side) subjects immediatelyfollowing the injection, and after a period of 5 hours.

FIG. 5A shows images of control (left side) and LPS-challenged (rightside) subjects in 5 minute increments in the first hour followinginjection of the particles. FIGS. 5B and 5C show measured luminescencefluxes (arbitrary units) from both lung and liver tissues as a functionof time, determined from the images in FIG. 5A. Measured luminescencefrom the lung tissue demonstrated that ROS were efficiently detectedcompared to the background signal corresponding to the control.Luminescence signals from liver tissue showed high background levels ofluminescence due to the presence in the liver of cytochrome P450oxidoreductases, which generate ROS.

To improve the selective targeting of lung tissues by the particles,particles were prepared as described above (e.g., with aminodextran anddextra aldehyde coatings, thioxene dye and chelated europium introducedinto the latex core) and conjugated to Ly6G antibodies, which bind tolung tissues. One group of BALB/cJ mice subjects was challenged with LPSintranasally with five 25 μL doses of 0.25 mg/mL LPS, followed by 25 μLof PBS. A control group of mice was challenged intranasally with five 25μL doses of PBS. Both groups of mice were allowed to wake up betweendoses. Five hours after the initial challenge, the mice were injectedwith the Ly6G-conjugated particles, and luminescence emission from theparticles was imaged.

FIG. 6A shows images of the control group (left) and the LPS-challengedgroup (right) immediately after injection of the particles, and after 5hours. FIG. 6B is a graph showing the relative measured luminescenceflux for both groups at both t=0 and t=5 hours. The measured fluxes att=5 hours show that detection of ROS in the lung tissues is readilyachieved by the particles.

To compare luminescence signals from lung and liver tissues form theLy6G-conjugated particles, BALB/cJ mice subjects were divided into twogroups. One group was challenged with LPS (four 25 μL doses of 0.25mg/mL followed by 25 μL PBS, delivered intratracheally) and the other(control) group was challenged with PBS (five 25 μL doses deliveredintratracheally). Five hours post-challenge, the mice were injectedintravenously with Ly6G1A8-conjugated particles (prepared as describedabove) and luminescence emission from the particles was imaged.

FIG. 7A shows images of the control and LPS-challenged groups at t=0 andt=5 hours following particle injection. FIG. 7B is a graph showing therelative contributions to the measured luminescence signals for thecontrol group (left bars) and LPS-challenged groups (right bars) formeasured signals from lung tissues, liver tissues, and the totalmeasured signal. For the control group, lung signals account for 28% ofthe measured luminescence, while liver signals account for the remaining72%. However, for the LPS-challenged group, lung signals account for 55%of the measured luminescence, while liver signals account for theremaining 45%. Both groups have similar total measured luminescencefluxes. These results show that by using antibody-conjugated particles,luminescence signals from ROS in specific tissues can be selectivelyinterrogated.

To investigate methods for further reducing luminescence signals due toliver tissues, two groups of BALB/cJ murine subjects were established.The first group was allowed normal access to food, while the secondfasted for approximately 23 hours. Control and LPS-challenged groupswere then constructed, with one fasting mouse and one mouse eatingnormally in each of the control and LPS-challenged groups. LPSchallenges were performed with four 25 μL doses of 0.25 mg/mL LPSdelivered intranasally, followed by 25 μL PBS. The mice of the controlgroup were challenged with PBS (five 25 μL doses deliveredintranasally). Five hours post-challenge, the mice were injectedintravenously with Ly6G1A8-conjugated particles (prepared as describedabove) and luminescence emission from the particles was imaged.

FIG. 8A shows images of the normal-eating and fasting mice in thecontrol and LPS-challenged groups as a function of time followinginjection of the particles. FIG. 8B is a graph showing contributions toeach of the measured luminescence signals (lung signal, liver signal,and total signal) from each of the different types of mice in each ofthe control groups. In each grouping of bars in FIG. 8B, thecontributions from left to right correspond to normal-eating controlgroup mice, fasting control group mice, normal eating LPS-challengedmice, and fasting LPS-challenged mice. In the control group, which themice ate normally, 87% of the measured luminescence signal wascontributed by liver tissues, while 13% was contributed by lung tissues.Where the mice fasted, however, 54% of the measured signal wascontributed by liver tissues, while 46% was contributed by lung tissues.Thus, in the control group, fasting significantly reduced theluminescence contributions from liver tissues, and is a viable methodfor improving the selective analysis of luminescence signals fromtissues in a subject's body other than liver tissues.

For the LPS-challenged group, fasting had little effect on the relativecontributions of liver and lung tissue-derived measured luminescencesignals. However, for the LPS-challenged mice, the conjugated particlesagain selectively targeted lung tissues relative to liver tissues.

To investigate the detection sensitivity of europium- and terbium-basedluminescent particles, a group of Swiss Webster mice were challengedwith LPS (1 mg/kg delivered subcutaneously through the right thigh). Theeuropium-based particles were prepared as described above. Terbium-basedparticles were prepared in a similar manner by substituting chelatedeuropium with chelated terbium.

Induction of ROS activity was monitored at five hours post-challengewith subcutaneous injection of 75 μg of the terbium particles in theleft thigh and 75 μg of the europium particles in the right thigh.Luminescence emission was imaged with no filter, with a 620 nm emissionfilter (the central wavelength of europium luminescence occurs at 614nm), and with a 540 nm emission filter (the central wavelength ofterbium emission occurs at 540 nm).

FIG. 9A shows a series of images of a murine subject before particleinjection, and after particle injection under the three differentfiltering arrangements. FIGS. 9B and 9C are graphs showingquantification of the Tb and Eu luminescence signals from the left andright thigh, respectively. At a wavelength of 540 nm, the Tb signallevel is only 6% of the total Tb luminescence signal (e.g., with nofilter), while at a wavelength of 620 nm, the Eu signal level is 65% ofthe total Eu luminescence signal (e.g., with no filter).

Other Embodiments

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe scope of the disclosure. Accordingly, other embodiments are withinthe scope of the following claims.

What is claimed is:
 1. A method, comprising: administering to a subjecta composition comprising particles, wherein each one of the particlescomprises at least one targeting group that binds to a structural entityin the subject and at least one reacting group that reacts chemicallywith a reactive oxygen species in the subject, and wherein the particleemits luminescence when the reaction occurs; detecting the luminescenceemission from the particles; and displaying an image of the subjectshowing locations of at least some reactive oxygen species in thesubject based on the detected luminescence.
 2. The method of claim 1,wherein administering the composition comprises injecting the particlesin a body of the subject.
 3. The method of claim 1, wherein the subjectis a living human.
 4. The method of claim 1, wherein the subject is aliving mammal.
 5. The method of claim 1, further comprising detectingluminescence emission at a wavelength of 500 nm or more.
 6. The methodof claim 1, further comprising detecting luminescence emission at awavelength of 600 nm or more.
 7. The method of claim 1, furthercomprising displaying the image of the subject based on unfilteredemitted radiation from the subject, wherein the unfiltered emittedradiation comprises the luminescence emission.
 8. The method of claim 1,wherein the at least one targeting group comprises at least oneantibody.
 9. The method of claim 1, wherein the reactive oxygen speciescomprises singlet oxygen.
 10. The method of claim 1, wherein thereactive oxygen species comprises hydroxide radical.
 11. The method ofclaim 1, wherein the reactive oxygen species comprises hypochlorousacid.
 12. The method of claim 1, wherein the reactive oxygen speciescomprises at least one member selected from the group consisting ofsuperoxide radical, nitric oxide, and hydrogen peroxide.
 13. The methodof claim 1, wherein each one of a first subset of the particlescomprises a first targeting group and a first reacting group, and eachone of a second subset of the particles comprises a second targetinggroup and a second reacting group, wherein the first and secondtargeting groups are different.
 14. The method of claim 13, wherein thefirst and second targeting groups bind to different structural entitiesin the subject.
 15. The method of claim 13, wherein the first and secondtargeting groups comprise different antibodies.
 16. The method of claim13, where each one of the first subset of the particles emitsluminescence at a first central wavelength, and each one of the secondsubset of the particles emits luminescence at a second centralwavelength different from the first central wavelength.
 17. The methodof claim 16, wherein each of the first and second central wavelengths isgreater than 500 nm.
 18. The method of claim 13, wherein the first andsecond reacting groups are the same.
 19. The method of claim 4, whereinthe mammal is a mouse.
 20. The method of claim 4, wherein the mammal isa rat.
 21. The method of claim 1, wherein the subject is selected fromthe group consisting of a living bird, a living amphibian, and a livingfish.
 22. A composition, comprising: a suspension medium; and aplurality of particles, wherein each one of the plurality of particlescomprises: a core comprising at least one reacting group that reactschemically with a reactive oxygen species in the subject, and at leastone luminescent agent that emits luminescence in response to in responseto the reaction of the at least one reacting group; a first coatingmaterial encapsulating the core; and a second coating materialencapsulating the first coating material and comprising at least onetargeting group that binds to a structural entity in the subject. 23.The composition of claim 22, wherein the core comprises at least one oflatex and polystyrene.
 24. The composition of claim 22, wherein thefirst coating material comprises aminodextran and the second coatingmaterial comprises dextran aldehyde.
 25. The composition of claim 22,wherein the at least one targeting group comprises at least oneantibody.
 26. The composition of claim 22, wherein the at least oneluminescent agent comprises at least one lanthanide element.
 27. Thecomposition of claim 26, wherein the at least one lanthanide elementcomprises at least one of europium and terbium.
 28. The composition ofclaim 22, wherein the at least one reacting group comprises thioxene ora thioxene derivative.
 29. The composition of claim 22, wherein theplurality of particles comprises: a first subset of particles comprisinga first targeting group, a first luminescent agent, and a first reactinggroup; and a second subset of particles comprising a second targetinggroup, a second luminescent agent, and a second reacting group, whereinthe first and second targeting groups are different.
 30. The compositionof claim 29, wherein the first and second targeting groups comprisedifferent antibodies.
 31. The composition of claim 29, wherein the firstand second luminescent agents are different.
 32. The composition ofclaim 31, wherein the first and second luminescent agents comprisedifferent lanthanide elements.
 33. A kit for imaging reactive oxygenspecies in a living subject, the kit comprising the composition of claim22.
 34. A kit for imaging reactive oxygen species in a living subject,the kit comprising the composition of claim
 29. 35. The kit of claim 33,wherein the reactive oxygen species comprises at least one memberselected from the group consisting of singlet oxygen, hydroxide radical,hypochlorous acid, superoxide radical, nitric oxide, and hydrogenperoxide.